earticle

영어

Quorum sensing (QS) is a cell density-dependent signaling system used by bacteria to coordinate gene expression within their population. In the QS mechanism of many gram-negative bacteria, acyl homoserine lactones (AHLs) are known to be the triggering molecules which form a complex with a transcriptional activator protein and promote the binding of the complex to DNA regulatory site thus activates the transcription of many virulence genes. In this study, we describe the development and characterization of in vivo (cell-based) and in vitro (protein-based) bioassay systems for detecting QS inhibitors for the three members of the LuxR family proteins, TraR, LasR and the recently identified QscR. For TraR and LasR, soluble proteins could be obtained from the cells which were grown in AHL-deficient medium but they did
not show binding affinity to the corresponding promoter sequences. Furthermore, the active LasR,
presumably produced as LasR-AHL complex, was not dissociated into its components (LasR and AHLs) in vitro. These findings indicate that the development of biochemical in vitro assay which requires pure and active TraR/LasR proteins might not be possible unless the structure of proteins and/or theirs folding processes are modified. Unlike the LasR and TraR, QscR could be obtained as an apo-protein that reversibly forms an active complex in vitro with its cognate signal molecule and, subsequently, binds to the target promoter DNA sequences. Both the in vitro assay and the in vivo assay using QscR-overproducing recombinant strains were applied for the screening process. Among furanones tested, several compounds showed strong and dose-dependent inhibitory effects against QS activity. These results suggest that (i) the in vivo and in vitro assay are equally sensitive and reliable tools for screening QS inhibitors, and (ii) furanones are potentially important QS inhibitors for many LuxR-type receptor proteins.